This invention generally relates to imaging objects or particles for purposes of detection and analysis, and more specifically, to a system and method for analyzing the three dimensional structure, contents, and spectral composition of objects, such as cells, which may be in motion.
There are a number of biological and medical procedures that are currently impractical due to limitations in cell and particle analysis technology. Examples of such procedures include battlefield detection and monitoring of both known and unknown toxins, non-invasive prenatal genetic testing and routine cancer screening via the detection and analysis of rare cells (i.e., cells having a low rate of occurrence) in peripheral blood, and drug discovery via high throughput cell assays.
New medical and biological procedures increasingly require more advanced cell analysis capabilities than currently exist. One example is the analysis of changes in the genetic constitution of tumor cells for the optimization of chemotherapy. Tumor cells may exhibit unusual DNA changes, such as a variation in the number of chromosomes, the amplification of chemotherapy-resistance genes, or changes in the regulation of gene expression. These changes can be detected using Fluorescence In-Situ Hybridization (FISH) probes that bind to specific DNA sequences within cells. FISH analysis requires an accurate determination of the number of distinct FISH locations within the nucleus of a cell, ideally in three dimensions. Commonly assigned U.S. patent application Ser. No. 09/490,478 describes the use of a stereoscopic imaging apparatus to view fluid-suspended cells from multiple angles, with a high numerical aperture for the accurate enumeration of FISH spots within a cell. This technique can be applied to a slide-mounted sample or a sample on a micro-fluidic chip, but generally with lower numerical apertures, due to the difficulty of coupling orthogonal collection systems to the flat sample substrate. Clearly, it would be preferable to perform three-dimensional (3D) imaging of cells on slides or in micro-capillaries with a single collection system in order to enable light collection with a high numerical aperture.
The most accurate determinations of FISH spot counts in cells on flat substrates are currently based on high-resolution fluorescence images taken at different focal planes across the depth of the cell. The resulting set of two-dimensional (2D) images are reconstructed into a three-dimensional (3D) representation of the cell, and FISH spots are counted both within and across the image planes to ensure that superimposed FISH spots within a single image are resolved across the multiple images. While such image stacking techniques can effectively resolve superimposed FISH spots, existing systems for 3D cell imaging are slow, often requiring several minutes to create each 3D composite. As a result, the various 2D images are gathered at widely different times, and changes in the cell over the course of the imaging process alter the resulting 3D representation. Further, such systems cannot tolerate movement of the cells during the imaging process, which limits their application to fixed cells that are immobilized on slides. An improved system would allow the rapid 3D imaging of cells, including cells in motion.
Accordingly, it will be apparent that an improved technique is desired that resolves the limitations in analyzing the three-dimensional features of both stationary and moving cells imposed by the conventional approaches discussed above. In addition, a new approach developed to address these problems in the prior art should also have application to the analysis of other types of objects besides cells and should be amenable to implementation in different configurations to meet the specific requirements of disparate applications of this technology.
The present invention is directed to an imaging method and system that is adapted to determine one or more characteristics of an object from an image of the object. There can be relative movement between the object and the imaging system, and although it is contemplated that either (or both) may be in motion, the object will preferably move while the imaging system is fixed in position. In addition, it should also be understood that while much of the following disclosure recites xe2x80x9can object,xe2x80x9d it is likely that the present invention will preferably be used with a plurality of objects and is particularly useful in connection with imaging a stream of objects or objects moving within a substrate, e.g., in narrow capillaries. Also, it should be understood that as used herein and in the following claims, the terms xe2x80x9cimagexe2x80x9d and xe2x80x9cimagingxe2x80x9d are broadly applied and are intended to generally refer to the light from an object or objects that is directed onto a surface of a detector; thus, these terms are intended to encompass light from an object or objects that is diffused, dispersed, or blurred on the surface of a detector, as well as light from an object or objects that is focussed onto the surface of the detector, and light from an object or objects that is divided into one or more spectral components incident on the surface of the detector.
The present invention may be applied to the rapid analysis of cells in three dimensions for purposes such as biological warfare agent detection, prenatal diagnosis, cancer screening, drug discovery, and other applications. To achieve such functional capabilities, the present invention collects image data from multiple focal positions within moving or stationary cells. Further, these data may be acquired over a large spectral range with high spectral and spatial resolution. The present invention preserves the spatial origin of the spectral information gathered from the object, enabling the discrimination of small sources of the same or different colored light emanating from the object. To accomplish these tasks, the present invention employs novel combinations of Time-Delay-Integration (TDI) imaging, synchronous and non-synchronous signal generation, and uses an optical system with an inclined detector orientation, and in some cases, a spectral dispersing element.
The TDI detector that is used in the various embodiments of the present invention preferably comprises a rectangular charge-coupled device (CCD) that employs various specialized pixel read out algorithms. Standard, non-TDI CCD arrays are commonly used for 2D imaging in cameras. In a standard CCD array, photons that are incident on a light sensitive element (corresponding to one pixel of an image and therefore referred to herein as a xe2x80x9cpixelxe2x80x9d) produce charges that are trapped in the element. After image acquisition, the photon charges from each light sensitive pixel are read into an output capacitor, producing a voltage proportional to the charge. Between pixel readings, the capacitor is discharged and the process is repeated for every pixel on the chip. During the readout, the array must be shielded from any light exposure to prevent charge generation in the pixels that have not yet been read.
In a TDI detector comprising a CCD array of physical pixels, the CCD array remains exposed to the light as the pixels are read out. The projection of an image on the array of physical pixels generates a pixilated signal. The readout typically occurs one row at a time, e.g., from the top to the bottom of the array. Once a first row is read out, the signal pixels in the remaining rows are shifted by one physical pixel in the direction of the row that has just been read. If the object being imaged onto the array moves in synchrony with the motion of the signal pixels, light from the object is integrated without image blurring for the duration of the TDI detector""s total readout period. The signal strength produced by a TDI detector increases linearly with the integration period, which is proportional to the number of physical TDI pixel rows, but the noise increases only as the square root of the integration period, resulting in an overall increase in the signal-to-noise ratio (SNR) compared to a conventional CCD array, by a factor equal to the square root of the number of rows.
In the present invention, there are four entities that may be in motion. These include the object being imaged, the image of the object projected on the detector, the detector itself, and the pixilated signal generated by the image on the detector. Any relative movement between the object and the detector results in movement of the image across the detector. TDI imaging, unlike other imaging methods, involves the movement of the pixilated signal across the detector while the measurement is being performed. In many cases, it is contemplated that the image of the object will move across the detector. However, the pixilated signal may not move in synchrony with the image of the object. In the present invention, the velocity of signal motion is a controllable parameter that can be adjusted in order to measure various features of the object being imaged. The signal can be made to move faster, slower, or in a different direction than the image, which may or may not itself be moving. Further, the movement of the signal can be changed dynamically during the measurement. The nature of the asynchrony in part determines the features of an object or objects that can be measured.
In TDI imaging of objects that was disclosed in previous pending applications, the image of the object moves synchronously with the pixilated signal (in the same direction and with the same speed), light forming each portion of the image is collected in the same portion of the pixilated signal over time, regardless of the motion. Conversely, if the image of the object moves asynchronously relative to the pixilated signal, (at a different speed and/or in a different direction), light forming each portion of the image at later times will not be collected in the same portion of the pixilated signal. By intentionally desynchronizing the motion of the pixilated signal on the TDI detector from the motion of the image, temporally distinct pixilated signals are produced. In the present invention, this effect, coupled with an inclination of the detector relative to the plane formed by the motion of the objects, can produce distinct images from different focal positions within the object. In the present invention, when the pixilated signal moves synchronously with the image, a characteristic signal intensity profile is produced that can be used to identify point sources of light such as those created by FISH probes.
Another adjustable parameter in the present invention is the continuity of signal generation. In some embodiments of the invention, the signal from the object is detected continuously. An exemplary application of continuous detection would be the imaging of a cell containing a chemiluminescent substrate that constantly emits light. Another example would be a cell illuminated by a continuous-wave laser or arc lamp to form a scatter, absorption, or fluorescence image on the detector. In other embodiments of the invention, the signal from the object is detected in a discontinuous fashion. For example, discontinuous detection would occur if a cell is illuminated by a pulsed or modulated laser to form transient scatter, absorption, or fluorescence images on the detector. Another example of discontinuous detection would be if a chemiluminescent cell is imaged via a shuttered or gated TDI detector. Signal continuity, when controlled in combination with the orientation of the detector plane and the synchrony of signal readout, gives rise to numerous embodiments and modes of operation of the present invention.